Epstein-Barr virus (EBV) is a herpesvirus which is associated with several malignancies. A central aspect of the life cycle and pathogenesis of EBV is a latent state of infection in which the viral chromosome is maintained as an episome. A single EBV- encoded protein, EBNA-1, allows the EBV chromosome to be maintained by binding to oriP, where it directs DNA replication to initiate and where it also acts to prevent the EBV chromosome from being lost from mitotically active cells. The replication function of oriP appears to be essential for the infection of primary B cells, although it is not needed when EBV infects established cell lines in culture. Molecular and genetic studies are proposed to investigate how EBNA-1 and cellular factors direct replication to initiate at oriP. Four specific aims are described: (1) We have evidence that in order to support replication initiation, two dimers of EBNA-1 must form a precise structure at oriP that involves a large DNA bend. We will investigate the angle of bending and whether a specific geometry is required. (2) We will take advantage of a wealth of information about DNA replication that has emerged from studies of yeast and Xenopus to begin to investigate the human proteins that may control initiation of DNA replication at oriP, using chromatin immunopreciptation and in vivo footprinting assays. (3) We will isolate replication-defective mutants of EBNA-1 that retain all other EBNA-1 activities and, therefore, may fail to perform an initiation step, such as interact with a host factor. (4) We will determine precisely where replication initiates at oriP by mapping the 5' ends of leading nascent strands. Each of these independent and complementary approaches should provide key insight into how replication initiates at oriP. The studies will also to be relevant to our understanding the replication of human chromosomes.